THERAPEUTIC AND DIAGNOSTIC VHH ANTIBODIES AGAINST SARS-COV-2 AND METHODS FOR THEIR ENHANCEMENT

Abstract

The present invention pertains in the fields of antibody technology, protein engineering, medicine, pharmacology, infection biology, virology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that prevent cell entry of and infection by SARS-CoV-2, a strategy for an enhanced block of the homotrimeric viral spike proteins by symmetry-matching VHH-fusions, implementations of this strategy, as well as VHH antibodies for sensitive detection of SARS-CoV2-infections.

Claims

1. A VHH antibody recognizing the SARS-CoV-2 spike protein 51 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain comprising (a) a CDR3 sequence as shown in SEQ. ID NO: 20, 204, 208, 4, 8, 12, 16, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, or 291, (b) a CDR3 sequence which has an identity of at least 80%, at least 90%, or at least 95% to a CDR3 sequence of (a), or (c) a VHH antibody, which competes with a VHH antibody of (a) for the binding to the SARS-CoV-2 spike protein 51 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

2. The VHH antibody according to claim 1 comprising (a) a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ. ID NO: 18-20, 202-204, 206-208, 2-4, 6-8, 10-12, 14-16, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-136, 138-140, 142-144, 146-148, 150-152, 154-156, 158-160, 162-164, 166-168, 170-172, 174-176, 178-180, 182-184, 186-188, 190-192, 194-196, 198-200, 225-227, 229-231, 233-235, 237-239, 241-243, 245-247, 249-251, 253-255, 257-259, 261-263, 265-267, 269-271, 273-275, 277-279, 281-283, 285-287, or 289-291, (b) a combination of CDR1, CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90%, or at least 95% to a combination of CDR1, CDR2 and CDR3 sequences of (a), or (c) a VHH antibody, which competes with a VHH antibody of (a) for the binding to the SARS-CoV-2 spike protein 51 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

3. The VHH antibody according to claim 1 comprising (a) a VHH sequence as shown in SEQ. ID NO: 17, 201, 205, 1, 5, 9, 13, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284 or 288, (b) a sequence which has an identity of at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% to a VHH sequence of (a), or (c) VHH antibody, which competes with a VHH antibody of (a) for the binding to the SARS-CoV-2 spike protein 51 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

4. The VHH antibody of claim 1, which recognizes the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

5. The VHH antibody of claim 1, which is capable of virus neutralization.

6. The VHH antibody of claim 1, which is capable of neutralizing a SARS-CoV2 mutant, in particular a SARS-CoV-2 escape mutant including the British mutant (Alpha), the South African mutant (Beta), Brazilian mutant (Gamma), the Indian mutant (Delta), the Californian mutant (Epsilon) as well as mutants comprising at least one of the amino acid substitutions in any one of the above mutants.

7. The VHH antibody of claim 1, which is capable of neutralizing a SARS-CoV2 mutant comprising a spike protein RBD including at least one amino acid substitution in the RBD selected from the group consisting of K417T, K417N, L452R, E484K, N501 and T478K.

8. The VHH antibody of claim 5, which neutralizes SARS-CoV-2 or a SARS-CoV-2 mutant at a concentration of about 500 pM or less, of about 250 pM or less, of about 170 pM or less, of about 100 pM or less, or of about 50 pM or less.

9. The VHH antibody of claim 1, which is stable, particularly thermostable or hyperthermostable.

10. The VHH antibody of claim 9, which has a melting temperature of at least about 65° C., of at least about 80° C., of at least 90° C. or of at least about 95° C. when measured under non-reducing conditions and/or under reducing conditions.

11. The VHH antibody of claim 9, which has an aggregation temperature of at least about 50° C., of at least about 60° C., of at least 70° C. or of at least about 80° C. when measured under non-reducing conditions and/or under reducing conditions.

12. The VHH antibody of claim 1, which is selected from antibody Re5D06 comprising a VHH sequence as shown in SEQ: ID NO. 17 or a VHH antibody, which is a variant thereof, particularly a variant comprising at least one of the mutations: A26I, I29S, I36M, Q41 E, S51A, I53W, S55N, S56N, T60V, N61D, N79D, V81Y, K89E, V95D, Y106X1 (with X1 being an amino acid residue selected from D, N, or E), 5108X2 (with X2 being any amino acid residue except for C), 5109X3 (with X3 being any amino acid residue, in particular E, Q or K, except for C or P), and Y111H, and more particularly a variant as shown in SEQ. ID NO: 201, (Re5D06R11), 205 (Re5D06R13), 224 (Re5D06R15), 228 (Re5D06R23), 232 (Re5D06R28), 236 (Re5D06R28D), 240 (Re5D06R15_3 QE), or 244 (Re5D06R28_3 QE).

13. The VHH antibody of claim 1, which is selected from antibody Re9H03 comprising a VHH sequence as shown in SEQ. ID NO: 252 or a VHH antibody, which is a variant thereof, particularly a variant as shown in SEQ. ID NO: 272 (Re22E05).

14. The VHH antibody of claim 1, which is selected from antibody Re5F10 comprising a VHH sequence as shown in SEQ. ID NO: 29 or a VHH antibody, which is a variant thereof, particularly a variant as shown in SEQ. ID NO: 260 (Re21 D01), 284 (Re26E09), or 288 (Re26E11).

15. The VHH antibody of claim 1, which is selected from antibody Re21H01 comprising a VHH sequence as shown in SEQ. ID NO: 264 or a VHH antibody, which is a variant thereof.

16. The VHH antibody of claim 1, which is selected from antibody Re25H10 comprising a VHH sequence as shown in SEQ. ID NO: 276 or a VHH antibody, which is a variant thereof.

17. The VHH antibody of claim 1, which is selected from antibody Re6H06 comprising a VHH sequence as shown in SEQ. ID NO: 73 or a VHH antibody, which is a variant thereof, particularly a variant as shown in SEQ. ID NO: 280 (Re26D07).

18. The VHH antibody of claim 1, which is covalently or non-covalently conjugated to a heterologous moiety, e.g. a labeling group, a capture group or an effector group, wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.

19. The VHH antibody of claim 1, which is fused to a heterologous polypeptide moiety.

20. The VHH antibody of claim 19, wherein the heterologous polypeptide moiety is a multimerization module, e.g. dimerization, trimerization or tetramerization module.

21. The VHH antibody of claim 20, which is a homo-trimerized VHH antibody fused to a trimerization module, e.g. a collagen trimerization moiety, particularly a human collagen moiety, or a lung surfactant protein D moiety.

22. The VHH antibody of any of claim 20, which is fused to a heterologous polypeptide moiety directly or via a spacer, e.g. a spacer having a chain length of 1-50 amino acids, particularly selected from (i) Gly, Ser, Glu and/or Asp, or from (ii) Gly, Glu, Ser and Pro.

23. The VHH antibody of claim 1, which is non-glycosylated.

24. The VHH antibody of claim 1, which is produced in a bacterium, e.g. E. coli.

25. The VHH antibody of claim 1, which is produced in a yeast, e.g. Pichia pastoris.

26. The VHH antibody of claim 1, which is conjugated to one or several polymer moieties, preferably hydrophilic polymer moieties, such as polyethylene glycol (PEG).

27. A set of two or more different VHH antibodies of claim 1.

28. The set of claim 27, wherein the different VHH antibodies recognize different epitopes on the RBD, particularly non-overlapping epitopes on the RBD.

29. The VHH antibody of claim 1 or a set of two or more different VHH antibodies of claim 1 in combination with a carrier suitable for use in medicine.

30. A method for preventing or treating a disorder caused by and/or associated with an infection with SARS-CoV-2 or a SARS-CoV-2 escape mutant, comprising administering a VHH antibody of claim 1 or a set of two or more different VHH antibodies of claim 1 to a patient in need of such treatment.

31. The method according to claim 30, wherein said patient is a human subject.

32. A method for detecting SARS-CoV-2 virus, comprising contacting a patient sample with the VHH antibody of claim 1 or a set of two or more different VHH antibodies of claim 1, wherein said set of two or more different VHH antibodies is selected from the group consisting of (i) a set of at least two VHH antibodies recognizing different, particularly non-overlapping epitopes on the receptor binding domain (RBD), (ii) a set of at least two VHH antibodies comprising a set of capturing antibodies conjugated to a capturing moiety and a set of labeling antibodies conjugated to a labeling moiety, and (iii) a set of at least two VHH antibodies comprising a first set of labeling antibodies conjugated to a first labeling moiety, and a second set of labeling antibodies conjugated to a second labeling moiety, wherein the first labeling moiety is different from the second labeling moiety, wherein the first and the second labeling moieties are particularly selected from two spectrally different fluorescence labeling moieties.

33. The method according to claim 32, wherein said patient sample is a body fluid or tissue sample.

34. A method for detecting SARS-CoV-2 virus or variants thereof or viral components in a virus culture or in a genetically modified organism, comprising contacting said virus culture or genetically modified organism with the VHH antibody of claim 1 or a set of two or more different VHH antibodies of claim 1.

35. The use of claim 34 for monitoring, quantification and/or quality control during production of viruses or viral components.

36. A nucleic acid molecule encoding a VHH antibody according to claim 1, preferably in operative linkage with a heterologous expression control sequence.

37. A vector comprising a nucleic acid molecule according to claim 36.

38. A recombinant cell or non-human organism transformed or transfected with a nucleic acid molecule according to claim 36 or a vector comprising a nucleic acid molecule according to claim 36.

39. The cell or organism of claim 38, which is selected from a bacterium such as E. coli Bacillus sp., a unicellular eukaryotic organism, e.g. yeast such as Pichia pastoris, or Leishmania, an insect cell, a mammalian cell or a plant cell.

40. A method for recombinant production of a VHH antibody, comprising cultivating a cell or an organism transformed or transfected with a nucleic acid molecule according to claim 36 or a vector comprising a nucleic acid molecule according to claim 36 in a suitable medium and obtaining the VHH antibody from the cell or organism or from the medium.

41. The method of claim 40, comprising cultivating a yeast such as Pichia pastoris and obtaining the VHH antibody from the medium.

42. A method for preventing or treating a disorder caused by and/or associated with an infection with SARS-CoV-2, comprising administering an effective dose of the VHH antibody of claim 1 or a set of two or more different VHH antibodies of claim 1, to a subject in need thereof, particularly to a human subject.

Description

FIGURES

[0348] FIG. 1: Sequence alignment and highlighting of variable regions

[0349] FIG. 1 shows an alignment of VHH sequences from Table 1. Residues that deviate from the consensus are shown in colour. The three variable CDR regions are indicated.

[0350] FIG. 2: Staining of transfected cells transiently expressing the SARS-CoV-2 spike protein.

[0351] HeLa cells where transfected with a plasmid carrying the humanized coding sequence of the SARS-CoV-2 spike protein under the control of a CMV promoter. 36 hours post-transfection, cells were fixed for 5 minutes with 4% paraformaldehyde (PFA), permeabilized, blocked with 5% BSA, incubated with 30 nM VHH Re6D09 carrying two Alexa568 fluorophores per molecule, extensively washed and finally imaged with an LSM780 confocal laser scanning microscope using the 405 nm and 568 nm laser lines for excitation. Image shows the overlaid DAPI and 568 channels, detecting DNA and VHH antibody in blue and red, respectively. Note that the transfection efficiency was only ˜30%. The bright red signal corresponds to transfected cells, while non-transfected ones served as negative control.

[0352] FIG. 3: Fluorophore-labelled anti-SARS-CoV-2 VHH antibodies specifically detect the spike protein and assembling viruses in infected cells.

[0353] Vero E6 cells were infected by SARS-CoV-2 for three days, fixed for two hours with 4% paraformaldehyde, and stained as described above with 30 nM of the indicated Alexa488-labelled VHH antibodies. Imaging was with a standard epifluorescence microscope.

[0354] FIG. 4: SARS-CoV-2 neutralizing VHH antibodies.

[0355] At day 0, Vero E6 cells were inoculated with SARS-CoV-2, in the absence of VHHs or after a 60 min pre-incubation with the indicated VHH antibodies. Three days later, the virus-load increased ˜10 000-fold when the antibodies had been omitted (compare “inoculation” and “no VHH”). One VHH (Re7D02) had no effect. Two (Re7D05 and Re5C08) had a weak impact. Re6B06 inhibited in this experiment ˜300-fold. The other 18 VHH antibodies blocked viral infection completely. Quantitation of viral RNA in the supernatants of infected cells was by quantitative reverse transcription (RT) PCR as described previously (Stegmann et al., 2020). Note the Log10-scale of the figure.

[0356] FIG. 5: VHH Re5D06 neutralizes SARS-CoV-2 with extreme potency.

[0357] Virus neutralization was performed as in FIG. 4. (A) Cells were fixed with PFA and stained with DAPI (to visualize cell nuclei), with a cocktail of Atto488-labelled VHHs recognizing the RBD (RBD epitopes) and Atto565-labelled VHHs recognizing an S1 epitope outside the RBD (S1ΔRBD epitope) in order to visualize newly synthesized Spike protein in successfully infected cells. Images show confocal sections. Cells that are positive in the Atto488 and Atto656 channels are infected. Note that infection was completely prevented when the virus was pre-incubated with VHH Re5D06 at a concentration of ≥50 pM. (B) A replicate experiment with identical outcome. Bars depict analysis by quantitative RT PCR, showing that ≥50 pM VHH Re5D606 prevented replication of the viral RNA completely.

[0358] FIG. 6: Hyper-thermostable anti-SARS-CoV-2 VHH antibodies.

[0359] Indicated VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposed aromatic/hydrophobic residues, which then bind and enhance fluorescence of the added SYPRO orange dye. Assays were performed in a volume of 20 μl, at 1 mg/ml VHH concentration in 50 mM Tris/HCl, 300 mM NaCl (pH 8.0 at 20° C.) and 1×dye (diluted from a 5000× stock; Life Technologies). Three replicates of each sample were pipetted in a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® CB′ Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed onto a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated for 5 min at 25° C. and then the temperature was increased in 1° C. increments of 45 seconds to 95° C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak. Note that the super-neutralizing VHH Re5D06 melts already at 50° C., while the optimized Re5D06R13 version remained fully stable throughout 95° C., as did Re6H06 and Re6B06. Re5D06R13 and Re6B06 retained their hyper-thermostability even in the presence of disulphide-bond reducing DTT.

[0360] FIG. 7: Highly potent symmetry matching anti SARS-CoV-2 VHH antibodies.

[0361] (A) Scheme of a homotrimeric VHH fusion to match the C3 rotational symmetry of the Spike of SARS-CoV-2 and other viruses. (B) Comparison of neutralization potencies of ReB06 monomers and Re6B06-spacer-Collagen XVIII NC1 trimers. The experiment was performed analogously to FIG. 5; however, overlaid fluorescent channels in extended focus are shown. Note that the trimer neutralizes at a 30,000-fold lower VHH concentration than the monomer. (C) Comparison between a VHH-72 monomer and a VHH-72 trimer. Note that the trimer neutralizes at a 10 000-fold lower VHH concentration than the corresponding monomer.

[0362] FIG. 8: Trimerization caused a strong avidity effect for the VHH-spike interaction

[0363] Hela cells were transfected as described in FIG. 2 to transiently express the SARS-Co-V2 spike protein. Following fixation, they were stained with Alexa488-labelled VHH Re6A11. In monomeric form, the staining was very weak even at 30 nM and with 2 fluorophores per VHH. In contrast, staining was strong with the trimerized version, even at a much lower concentration of 1 nM VHH and with only one fluorophore per VHH.

[0364] FIG. 9: Neutralization of SARS-CoV-2 B.1.351 by VHH antibodies.

[0365] (A) and (B): Neutralization of SARS-CoV-2 B.1.351 by the indicated VHH antibody constructs. The neutralization experiment was performed as described in FIG. 5, with the difference being that a mix of mutant-optimized anti-RBD/S1 VHH antibodies were used for immunofluorescence staining.

TABLE-US-00005 VHH antibody sequences >Re5A08 GSQVQLVESGGGLVQAGGSLRLSCTASGHTFTANRMGWFRQAPGKER EFVAAINWGGDSTNYVDSVKGRFTISRDIAKNTVYLQMNSLKPEDTA VYFCAARNHVTGEFDSWGQGTQVTVSSTS >Re5B06 GSQVQLVESGGGLVQPGGSLRLSCAASGSIRSIYATVWFRQAPGKEH EWVGSITSSNVTTYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAL YYCNVHFASEYSDYAQIQGQGTQVTVSSTS >Re5C01 GSQVQLVESGGGLVQAGGSLRLSCGVSGRTFSSYAMGWFRQAPGKER EFVATISWSGGTTNYAHSVKGRFTISRDNAKNTVYLQMNSLKVEDTA VYYCYAVSSGSDYDGGMDYWGKGTQVTVSSTS >Re5C08 GSQVQLVESGGGSVEAGGSLRLSCAASGRTFNDYNMVWFRQAPGKER EFVAAIKWNGGNTSYADSVKGRFAVSRDNAKNTVYLQMNNLKHEDTA EYLCYTVGPEGDYWGQGTQVTVSSTS >Re5D06 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAIGWFRQAPGKER EGVSRIRSSDGSTNYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTA VYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re5E03 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSCISNSDGSTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAGGPQTYYSGSYYYTCAEGAMDYWGKGTLVTVSSTS >Re5E11 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSCISSSDGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCATAPLTYYSGSWYLTCNSDAMDYWGKGTLVTVSSTS >Re5F10 GSQVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKEC EWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCRRGQGTQVTVSSTS >Re5F11 GSQVQLVESGGALVQPGGSLRLSCATSGSISSYRMGWYRQGPGKQRE LVAFITIGGITDYIDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVY YCNADPPLFNWGQGTQVTVSSTS >Re5G05 GSQVQLVESGGGLVQAGGSLRLSCAASGFTATSYAMGWYRQAPGKEC EWVATITSTGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCDWGQGTQVTVSSTS >Re6A11 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKER EFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YSCAAASDYGLPREDFLYDYWGQGTLVTVSSTS >Re6B02 GSQVQLVESGGALVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKER EGVSRIRSSDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re6B06 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFSSAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTS >Re6B07 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSYIRSSDGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAADEAYYSELGWESPWGWSYWGQGTRVTVSSTS >Re6D06 GSQVQLVESGGGLVQAGASLRLSCAASGRMFGVYRMGWFRQAPGKER EFVAGISTSVGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAARDPTTYEYDYWGQGTQVTVSSTS >Re6D08 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFHQAPGKER EFVATINWSGDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAAVVDPSPTYYSGKYYPPRVEYWGKGTQVTVSSTS >Re6D09 GSQVQLVESGGGSVEAGGSLRLSCAASGRTFNNYNMVWFRQAPGKER EFVAAINWNGGSTSYAASVKGRFAVSIDNAKNTLYLQMNNLKHEDTA EYLCYTVGPEGDYWGQGTQVTVSSTS >Re6E11 GSQVQLVESGGGLVQPGGSLRLSCAASGLTLDYYAIGWFRQAPGKER EGVSCISSRDGSTMYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAATPTTYYSGSYYYTCSPEGYDYWGQGTQVTVSSTS >Re6F06 GSQVQLVESGGGLVQAGGSLRLSCAASGFTFSNYAMGWYRQAPGKEC EFVAVITITGSNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCESQGQGTRVTVSSTS >Re6G03 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYRMAWFRLAPGKER EFVAGINWSDGTTSYKDSVKGRFTISRDNAKNTVYLQMDSLKPEDTA VYYCNAHLSTGQEGPGEYFGMDYWGKGTQVTVSSTS >Re6H06 GSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKER EGVSCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTG VYYCAVVPQTYYGGKYYSQCTANGMDYWGKGTLVTVSSTS >Re6H10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWYRQAPGKEC EFVAVITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCRGGGQGTLVTVSSTS >Re7A01 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKER EFVATISFSGSTSYAGHVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCHAVTRASDQDGGMDYWGQGTQVTVSSTS >Re7B01 GSQVQLVESGGGLVQPGGSLRLSCGASGFTLDYYAIGWFRQAPGKER EGVSRIRSNDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re7D05 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKER EFVATISWSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNAVTHHSDQDGGMDYWGKGTLVTVSSTS >Re7E02 GSQVQLVESGGGLVQAGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSYIRSSDGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAADEAYYSELGWESPWGWSYWGQGTQVTVSSTS >Re7H02 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAENTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTS >Re8A03 GSQVQLVESGGGLVQPGGSLRLSCAASGRITGFNGMGWYRQTPGKQR ELVASITNGGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YLCYFWRPEFPNLYWGQGTQVTVSSTS >Re8A06 GSQVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKER ELVAAMGSSGWINYADSVKGRLTISRDNAKNTLYLQMNSLKPEDTAV YYCRGTGGVGPTSADYWGQGTQVTVSSTS >Re8C06 GSQVQLVESGGGLVQAGGSLRLSCAASGRTDTIYNMGWFRQAPGKER EFVAAISWSDGKTTFADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA NYYCAAKAFLVAGRSLEEYDYSGQGTQVTVSSTS >Re8E12 GSQVQLVESGGGSVQPGGSLRLSCKVSGFTSDVDLRNYLVSWNRQAP GKERELVAAITPTVISGGNTNYADSVKGRFTISRDYSKSTVYLQMNS LNPEDTAVYYCKVGVYWGQGTQVTVSSTS >Re8F03 GSQVQLVESGGGLVQPGGSLTLSCKVSGLTSYVDLRNYLVSWYRQGP GKERELVAAITPTAITGGSTNYADSVKGRFTISRDYSKSTVYLQMNS LNPEDTAVYSCKVGVYWGQGTQVTVSSTS >Re9B09 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCATVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTS >Re9B10 GSQVQLVESGGGLVQPGGSLRLSCAASGRMFGVYRMGWFRQAPGKER EFVAGISTSVGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAARDPTTYEYDYWGQGTQVTVSSTS >Re9C07 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKER EFVAAITWNADTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAAGGNHYYSRSYYSSLEYDHWGQGTQVTVSSTS >Re9C08 GSQVQLVESGGGLVQPGGSLRLSCAVSGNIFGITAWDWHRQAPGKQR ELVAHITSRGDTYYLDSVKGRFAISRDHAKNTLSLQMNSLKPEDTAV YYCYLRTFGPPNDHWGQGTQVTVSSTS >Re9D02 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKER EFVAAISWGGDTTYYADSLKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAADRGLSYYYDRVTEYDYWGQGTQVTVSSTS >Re9G05 GSQVQLVESGGGLVQPGGSLRLSCAVSGNISSITAWDWHRQAPGKQR ELVAHITSRGDTMYLDSVKGRFAISRDHAKNTLSLQMNSLKPEDTAV YYCYLRTFGPPYDYWGQGTQVTVSSTS >Re9G12 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKER EFVAHISWSGDSTYYADSVKGRFTIFRDNAKNTAYLQMNSLKPEDTA VYYCAADRGASYYYTWASEYNYWGQGTQVTVSSTS >Re9H01 GSQVQLVESGGGLVQAGDSLRLSCAASGNIFSINAMGWYRQAPGKQR ELVAFITSRGSTNYTDSVKGRFTISRDTAKDTVYLQMNSLKPEDTAV YFCRGGYSDYDIYFGSWGQGTQVTVSSTS >Re10B02 GSQVQLVESGGGLVQPGGSLRLSCATSGSISSYRMGWYRQGPGKQRE LVAFITIGGITDYIDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVY YCNADPPLFNWGQGTQVTVSSTS >Re10B10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRIRSSDGSTTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re10F10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRIRNNDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re11C10 GSQVQLVESGGGSVEAGGSLRLSCAASGRTLDNYNAVWFRQAPGKER EFVAAINWNGSNTSYGNSVKGRFAVSRDNAKNTVYLQMNNLKHEDTA EYLCYTVGPEGDYWGQGTQVTVSSTS >Re11E11 GSQVQLVESGGGSVEAGGSLRLSCAASGRTFNNYNIVWFRQAPGKER EFVAAINWNGGSTSYANSVKGRFAVSRDNAKNTVYLQMNNLKHEDTA EYLCYTVGPEGDYWGQGTQVTVSSTS >Re11F07 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRQAPGKER EFVATISWSGGSTSYARSVKGRFTISGDNAENTVYLQMNSLKPEDTA VYYCYAVSSGSDYDGGMDYWGKGTLVTVSSTS >Re11F11 GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSNYHMSWYRQAPGKGR ELVADITSGGDYTHYADSVKGRFTVSRDNPKNTLYLQMNSLKPEDTA VYHCHVRIFGPGFPVDYRGQGTQVTVSSTS >Re11G09 GSQVQLVESGGGLVHTGGSLRLSCAASGSIFNIYRMAWYRQAPGKQR EKVAIITTYGLTDYADSVKGRFTISRDNAKNTTYLQMNSLKPDDTAV YYCNTDPPDLGPGYWGQGTQVTVSSTS >Re11H04 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYTIAWFRQAPGKER EGVSCISGNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAADRGESYYPFRPSEYHYWGQGTQVTVSSTS >KG4B11 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTS >Re5D06R11 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAMGWFREAPGKER EGVARIRSNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA VYYCAYGPLTKYGSEWYWPYEYDYWGQGTQVTVSSTS >Re5D06R13 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAMGWFREAPGKER EGVARIRSNDGSVNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA DYYCAYGPLTKYGSEWYWPYEYDYWGQGTQVTVSSTS >Re5D06R15 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAMGWFREAPGKER EGVARIRNSDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA DYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re5D06R23 GSQVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKER EGVARIRNNDGSTDYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA DYYCAYGPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re5D06R28 >GSQVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKE REGVARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDT ADYYCAYGPLTKYGSSWHWPYEYDYWGQGTQVTVSSTS >Re5D06R28D GSQVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKER EGVARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA DYYCAYGPLTKYGDEWHWPYEYDYWGQGTQVTVSSTS >Re5D06R15_3QE GSQVQLVESGGGLVEPGGSLRLSCAASGITLDYYAMGWFREAPGKER EGVARIRNSDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA DYYCAYGPLTKYGSSWYWPYEYDYWGEGTEVTVSSTS >Re5D06R28_3QE GSQVQLVESGGGLVEPGGSLRLSCAISGSTLDYYAMGWFREAPGKER EGVARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTA DYYCAYGPLTKYGSSWHWPYEYDYWGEGTEVTVSSTS >Re9F06 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKER EFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YSCAAASDYGLPREDFLYDYWGQGTQVTVSSTS >Re9H03 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCATVPGTYYSGNWYYTWHPKAVDYWGKGTLVTVSSTS >Re21B09 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDNYAIGWFRQAPGKER EGVSCIRSSDGSTYYADSVKGRFTISKDNAKNTVYLQMNSLKPEDTA VYYCATDGTFNPPCDDLYSWYFPERQGTQVTVSSTS >Re21D01 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKEC EWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCRRGQGTQVTVSSTS >Re21H01 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKEC EWVATITITGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCNPDPGCRGGGQGTQVTVSSTS >Re22D04 GSQVQLVESGGGLVQTGGSLRLSCAASGRTFSDDAMGWFRQAPGKER DVVAALGWAGVSTYYADSVKGRFGISRDNAKNTVYLQMSSLKPEDTA VYYCAAAPSVAHARLGEWAYWGKGTQVTVSSTS >Re22E05 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCATVPGTYYSGNWYYTWHPKAVDYWGKGTQVTVSSTS >Re25H10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMSWARQAPGKGL EWVSTISEDGSTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAA YYCATSTEPRTVVAGWGDYLGQGTQVTVSSTS >Re26D07 GSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKER EGVSCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTG VYYCAVVPQTYYGGKYYSQCTANGMDYWGKGTQVTVSSTS >Re26E09 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKEC EWVATITITGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCRRGQGTRVTVSSTS >Re26E11 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKEC EWVATITITGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCNPDPGCRRGQGTQVTVSSTS >VHH-72 monomer GSGQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDT AVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGS Sequences of heterodimeric VHH antibodies >Re9F06-SpacerA-Re9B09|pDG03599 (SEQ. ID NO: 292) GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKER EFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGEGGEGSQV QLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVS RISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC ATVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTS >Re9F06-SpacerA-Re5D06R28D|pDG03560 (SEQ. ID NO: 293) GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKER EFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGEGGEGSQV QLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVA RWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYC AYGPLTKYGDEWHWPYEYDYWGQGTQVTVSSTS >Re22D04-SpacerA-Re5D06R28D|pDOG3661 (SEQ. ID NO: 294) GSQVQLVESGGGLVQTGGSLRLSCAASGRTFSDDAMGWFRQAPGKER DVVAALGWAGVSTYYADSVKGRFGISRDNAKNTVYLQMSSLKPEDTA VYYCAAAPSVAHARLGEWAYWGKGTQVTVSSTSGEGEGGEGGEGSQV QLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVA RWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYC AYGPLTKYGDEWHWPYEYDYWGQGTQVTVSSTS >Re25H10-SpacerA-Re9B09|pDG03697 (SEQ. ID NO: 295) GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMSWARQAPGKGL EWVSTISEDGSTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAA YYCATSTEPRTVVAGWGDYLGQGTQVTVSSTSGEGEGGEGGEGSQVQ LVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSR ISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA TVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTS >Re25H10-SpacerA-Re5D06R28D|pDG03798 (SEQ. ID NO: 296) GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMSWARQAPGKGL EWVSTISEDGSTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAA YYCATSTEPRTVVAGWGDYLGQGTQVTVSSTSGEGEGGEGGEGSQVQ LVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVAR WRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCA YGPLTKYGDEWHWPYEYDYWGQGTQVTVSSTS >Pp086|Re9F06-SpacerB-Re5D06R28D|pDG03637 (SEQ. ID NO: 297) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGS QVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREG VARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADY YCAYGPLTKYGDEWHWPYEYDYWGQGTQVTVSS >Pp087|Re9F06-SpacerC-Re6H06|pDG03625 (SEQ. ID NO: 298) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGSGE GGSEGGEGGSGEGSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYA IGWFRQAPGKEREGVSCTSSSDGSTYYADSVKGRFTISRDNAKNTVY LQMNSLKPEDTGVYYCAVVPQTYYGGKYYSQCTANGMDYWGKGTLVT VSS >Pp088|Re9F06-SpacerC-Re9B09|pDG03626 (SEQ. ID NO: 299) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGSGE GGSEGGEGGSGEGSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYA IGWFRQAPGKEREGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCATVPGTYYSGNWYYTWHPEAVDYWGKGTQVT VSS >Pp089|Re9F06-SpacerB-Re6H06|pDG03627 (SEQ. ID NO: 300) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGS QVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREG VSCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVY YCAVVPQTYYGGKYYSQCTANGMDYWGKGTLVTVSS >Pp090|Re9F06-SpacerB-Re9B09|pDG03628 (SEQ. ID NO: 301) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGS QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREG VSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCATVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSS >Pp091|Re9F06-SpacerB-Re5D06R15_3QE|pDG03629 (SEQ. ID NO: 302) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGS QVQLVESGGGLVEPGGSLRLSCAASGITLDYYAMGWFREAPGKEREG VARIRNSDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADY YCAYGPLTKYGSSWYWPYEYDYWGEGTEVTVSS >Pp092|Re9F06-SpacerB-Re5D06R28_3QE|pDG03630 (SEQ. ID NO: 303) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKE REFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYSCAAASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGS QVQLVESGGGLVEPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREG VARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADY YCAYGPLTKYGSSWHWPYEYDYWGEGTEVTVSS >Pp093|Re21D01-SpacerB-Re5D06R15_3QE|pDG03663 (SEQ. ID NO: 304) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVEPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGVARIRNSDG STNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTK YGSSWYWPYEYDYWGEGTEVTVSS >Pp094|Re21D01-SpacerB-Re5D06R28_3QE|pDG03664 (SEQ. ID NO: 305) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVEPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDG STNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTK YGSSWHWPYEYDYWGEGTEVTVSS >Pp095|Re21D01-SpacerC-Re5D06R15_3QE|pDG03665 (SEQ. ID NO: 306) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGG SGEGSQVQLVESGGGLVEPGGSLRLSCAASGITLDYYAMGWFREAPG KEREGVARIRNSDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPE DTADYYCAYGPLTKYGSSWYWPYEYDYWGEGTEVTVSS >Pp096|Re21D01-SpacerC-Re5D06R28_3QE|pDG03666 (SEQ. ID NO: 307) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGG SGEGSQVQLVESGGGLVEPGGSLRLSCAISGSTLDYYAMGWFREAPG KEREGVARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPE DTADYYCAYGPLTKYGSSWHWPYEYDYWGEGTEVTVSS >Pp097|Re21D01-SpacerB-Re6H06|pDG03667 (SEQ. ID NO:308) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGVSCTSSSDG STYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAVVPQTY YGGKYYSQCTANGMDYWGKGTLVTVSS >Pp098|Re21D01-SpacerB-Re9B09|pDG03668 (SEQ. ID NO: 309) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDG STDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTY YSGNWYYTWHPEAVDYWGKGTQVTVSS >Pp099|Re21D01-SpacerC-Re6H06|pDG03669 (SEQ. ID NO: 310) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGG SGEGSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPG KEREGVSCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTGVYYCAVVPQTYYGGKYYSQCTANGMDYWGKGTLVTVSS >Pp100|Re21D01-SpacerC-Re9B09|pDG03670 (SEQ. ID NO: 311) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKE CEWVATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNPDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGG SGEGSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPG KEREGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCATVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSS Sequences of subunits for trimeric VHH-spacer Collagen XVIII NC1 or collagen XV NC1 fusions expressed in E. coli >Re6B06 ColXVIII trimer (SEQ. ID NO: 211) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFSSAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGAD ASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQ NGFRKVQLEARTPLPR >Re7H02 ColXVIII trimer (SEQ. ID NO: 212) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAENTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGAD ASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQ NGFRKVQLEARTPLPR >KG4B11 ColXVIII trimer (SEQ. ID NO: 213) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGAD ASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQ NGFRKVQLEARTPLPR >Re6D06 ColXVIII trimer (SEQ. ID NO: 214) GSQVQLVESGGGLVQAGASLRLSCAASGRMFGVYRMGWFRQAPGKER EFVAGISTSVGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAARDPTTYEYDYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPG EQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQE ELYVRVQNGFRKVQLEARTPLPR >Re6A11/Re9F06 ColXVIII trimer (SEQ. ID NO: 215) GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKER EFVAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YSCAAASDYGLPREDFLYDYWGQGTLVTVSSTSEGSEGPESSDGSDS TDPGEQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFV AEQEELYVRVQNGFRKVQLEARTPLPR >Re5A08 ColXVIII trimer (SEQ. ID NO: 216) GSQVQLVESGGGLVQAGGSLRLSCTASGHTFTANRMGWFRQAPGKER EFVAAINWGGDSTNYVDSVKGRFTISRDIAKNTVYLQMNSLKPEDTA VYFCAARNHVTGEFDSWGQGTQVTVSSTSEGSEGPESSDGSDSTDPG EQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQE ELYVRVQNGFRKVQLEARTPLPR >VHH-72 ColXVIII trimer (SEQ. ID NO: 218) GSGQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDT AVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGSGSEGPESSDGS DSTDPGEQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLI FVAEQEELYVRVQNGFRKVQLEARTPLPR > ColXV-Re9B09 ColXV trimer (SEQ. ID NO: 315) GSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKK LQLGELIPIPAGSEGPESSDGSDSTDPGEQGEGADASDGSEGGSQVQ LVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSR ISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA TVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTS >Re9B09-ColXVIII trimer (SEQ. ID NO: 316) GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCATVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTSEGSEGPE SSDGSDSTDPGEQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVP EGWLIFVAEQEELYVRVQNGFRKVQLEARTPLPR > ColXV-Re9H01 trimer (SEQ. ID NO: 317) GSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKK LQLGELIPIPAGSEGPESSDGSDSTDPGEQGEGADASDGSEGGSQVQ LVESGGGLVQAGDSLRLSCAASGNIFSINAMGWYRQAPGKQRELVAF ITSRGSTNYTDSVKGRFTISRDTAKDTVYLQMNSLKPEDTAVYFCRG GYSDYDIYFGSWGQGTQVTVSST >Re9H01-ColXVIII trimer (SEQ. ID NO: 318) GSQVQLVESGGGLVQAGDSLRLSCAASGNIFSINAMGWYRQAPGKQR ELVAFITSRGSTNYTDSVKGRFTISRDTAKDTVYLQMNSLKPEDTAV YFCRGGYSDYDIYFGSWGQGTQVTVSSTSEGSEGPESSDGSDSTDPG EQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQE ELYVRVQNGFRKVQLEARTPLPR > ColXV-Re7H02 trimer (SEQ. ID NO: 319) GSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKK LQLGELIPIPAGSEGPESSDGSDSTDPGEQGEGADASDGSEGGSQVQ LVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFVAS VSWSGDSTNYADSVKGRFTISRDNAENTGYLQMNSLKPEDTAVYYCK RGPYWGQGTQVTVSSTS > KG4B11-ColXV trimer (SEQ. ID NO: 320) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKER EFVASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTA VYYCKRGPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGAD ASDGSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDG WKKLQLGELIPIPAD Ost1 signal peptides >S. cerevisiae Ost1 signal peptide (SEQ. ID NO: 219) MRQVWFSWIVGLFLCFFNVSSAA >P. pastoris Ost1 signal peptide (SEQ. ID NO: 220) MKFISILFLLIGSVFG >S. pombe Ost1 signal peptide (SEQ. ID NO: 221) MLVLKLLLWSIISGLSLAE >C. albicans Ost1 signal peptide (SEQ. ID NO: 222) MWKFFITLGVIFSICSA Propeptide sequence >S. cerevisiae propeptide alpha factor (SEQ. ID NO: 223) APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLL FINTTIASIAAKEEGVSLEKR

EXAMPLES

Example 1—Periplasmic Expression and Purification of VHH Antibody Monomers

[0366] VHH antibody Re6H06 was expressed with an N-terminal pelB signal sequence and a C-terminal His10 tag from a Kan-ColE1 μlasmid harboring a T5/lac promoter in E. coli NEBExpress (New England Biolabs). A 125-ml pre-culture in Terrific Broth (TB) containing 50 μg/ml Kanamycin was grown overnight at 28° C. to early stationary phase. The culture was then diluted with fresh medium (500 ml, pre-warmed to 37° C.). After 30 minutes of growth at 37° C., protein expression was induced with 0.05 mM IPTG and growth was continued for 2 hours at 37° C., whereby the culture reached a final OD.sub.600 of ˜8. Bacteria were harvested by centrifugation and lysed by osmotic shock lysis: cell pellets were resuspended in 14 ml 130 mM Tris/HCl pH 8.0, 10 mM EDTA, and sucrose was immediately added to 20% (w/v). After gentle mixing at 23° C. for 30 min, four volumes of ice-cold water were added and mixing was continued at 4° C. for 30 min. 20 mM Tris/HCl pH 7.5, 50 mM NaCl and 20 mM imidazole were added to the cell suspension. Periplasmic extract was then recovered as the supernatant of two consecutive centrifugation steps at 4° C.: a low-speed spin at 4000 xg (20 min, F13 rotor, Thermo Fisher Scientific) and a high-speed spin at 38000 rpm (˜1 hour, T647.5 rotor). The VHH antibody was purified at 4° C. via Ni.sup.2+ EDTA-amide chelate affinity chromatography (1 ml matrix). Beads were washed with ten column volumes of 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.2% (w/v) Triton X-100 and ten column volumes of buffer lacking detergent. After elution with 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 500 mM imidazole, the buffer was exchanged to 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 250 mM sucrose via a PD 10 desalting column (GE Healthcare). Aliquots were frozen in liquid nitrogen and stored at −80° C. This expression/purification strategy was used for all VHH antibodies containing four cysteines and thus two disulfide bonds (Re5E03, Re5E11, Re5G05, Re6E11, Re6F06, Re6G03, Re6H06, Re6H10) as well as for Re5F11.

Example 2—Cytoplasmic Expression and Purification of VHH Monomers

[0367] All other VHH antibodies comprising just two cysteines and thus a single disulfide bond were produced as His14-ScSUMO fusions by cytoplasmic expression in E. coli NEBExpress Shuffle, which allows forming disulfide bonds in the bacterial cytoplasm. In brief, 125 ml pre-cultures were grown overnight at 35° C. in TB+50 μg/ml kanamycin in 5 liter flasks to early stationary phase. They were then diluted with 250 ml fresh medium, shifted to 21° C. and induced for 5 hours with 0.08 mM IPTG. 5 mM EDTA was added, bacteria were pelleted, resuspended in 50 mM Tris/HCl pH 7.5, 20 mM imidazole/HCl pH 7.5, 300 mM NaCl, frozen in liquid nitrogen and lysed by thawing plus sonication. Insoluble material was removed by ultracentrifugation at 38000 rpm (˜1 hour, T647.5 rotor).

[0368] The supernatant was applied to a 1 ml Ni.sup.2+ EDTA-amide chelate column. The matrix was sequentially washed in resuspension buffer, resuspension buffer+0.2% TritonX100, resuspension buffer+700 mM NaCl, low salt buffer (resuspension buffer minus NaCl), and protease buffer (resuspension buffer with an imidazole concentration lowered to 10 mM and supplemented with 250 mM sucrose). The VHH antibody was finally eluted by cleaving the His14-SUMO-tag using 100 nM S. cerevisiae Ulp1 for 2 hours at room temperature. The eluted VHH antibodies were frozen in liquid nitrogen and stored an −80° C. until further use.

Example 3—Cytoplasmic Expression and Purification of VHH Trimers

[0369] The VHH trimers listed in Table 4 were expressed and purified as described for the VHH monomers, the only difference being that the VHH sequence was C-terminally extended by a 31 residues long spacer (in single letter code: EGSEGPESSDGSDSTDPGEQGEGADASDGSE) (SEQ. ID NO: 209) and by the 57 residues long human collagen XVIII NC trimerization domain

TABLE-US-00006 (SEQ. ID NO: 210) (in single letter code:  GASSGVRLWATRQAMLGQVHEVPEGWLIF VAEQEELYVRVQNGFRKVQLEARTPLPR).

Example 4—Spike Protein-Staining in Transfected Cells

[0370] To prepare fluorophore-labelled probes, monomeric VHH antibodies were expressed with two additional cysteine residues, one at the N- and one at the C-terminus. These were then used for labelling with fluorophore-maleimides as described by Pleiner et al., 2015. The trimerized VHH antibody Re6A11 was labelled through a single N-terminal cysteine. Labelings were essentially quantitative as judged by ratiometric UV-VIS spectroscopy and electrophoretic size shifts.

[0371] HeLa cells were cultivated in DMEM+ 5% FCS, seeded in 10-well slides, and transiently transfected with a plasmid allowing expression of the SARS-CoV-2 spike protein from a CMV promoter and using the PolyJet transfection reagent according to the manufacturer's instruction (SignaGen). 2 days later, cells were fixed for 5 minutes with 4% paraformaldehyde (PFA) in PBS, washed in PBS, permeabilized with 0.5% saponin, blocked with 5% BSA in PBS, and incubated with gentle shaking for 60 min with fluorophore-labelled VHH antibodies (in PBS+ 1% BSA) at concentrations given in the figures. Following washes with PBS and counterstaining with DAPI, images were taken by confocal laser scanning microscopy.

Example 5—Spike Protein Detection in SARS-CoV-2 Infected Cells

[0372] Stainings were performed with Vero E6 cells infected for 2 days as described above. Fixation was with 4% paraformaldehyde (PFA) for 2 hours. This long period of fixation was due to safety reasons to make sure that no infectious material remained. As a consequence, only highly fixation-resistant epitopes remain visible—explaining the difference between the staining of transfected and virus-infected cells in Table 1. The fluorescence staining themselves were performed as described above. FIG. 3 shows epifluorescence images. The evaluations of Table 1 are based on confocal laser scans.

[0373] Example 6—Virus Infection and Neutralization Assays

[0374] Virus stocks were prepared as supernatants from Vero E6 cells infected with SARS-CoV-2. The virus stocks contained between 10.sup.11 and 10.sup.12 copies of the virus genome per ml, as determined by reverse transcription and quantitative PCR (qRT-PCR) according to a standard protocol (Corman et al., 2020). 2*106 virus copies were diluted in 100 μl of cell culture media, DMEM/2% FBS, in the presence or absence of varying concentrations of the VHH antibodies under study. After 60 min incubation at 37° C., this was added to a monolayer of Vero E6 cells, i.e. 5000 cells in a well of a standard 96 well cell culture plate. After 48 or 72 hours of incubation, the supernatants of the cells were harvested. To quantify the amount of newly produced virus particles, the RNA from this material was isolated by the Trizol method, followed by qRT-PCR. Alternatively, cells were fixed as in Example 5, stained with a cocktail Atto488 labelled anti-RBD VHHs (10-15 nM each) and with 15 nM Atto565-labelled VHH (Re8H11) that recognizes an S1 SARS-CoV-2 epitope outside the RBD. Imaging was by confocal laser scanning or spinning disc fluorescence microscopy. Neutralization was considered as successful if no infected cells were detectable within the well.

[0375] Example 7—Producing Trimeric VHH Antibodies by Secretion from Pichia pastoris

[0376] The sequences of VHH trimers were cloned into a vector that was derived from pPICZα (Invitrogen/ThermoFisher Scientific, Cat. No. V19520) by replacing the signal peptide of the mating factor α precursor by the signal peptide of ScOst1p (Barrero et al., 2018) and replacing the zeocin resistance cassette by a coding sequence for the aminoglycoside 3′ phosphotransferase (UniProt ID: KKA1_ECOLX), conferring resistance to kanamycin and G418. The plasmids encoding VHH antibody trimers were transformed into wild-type P. pastoris cells using the protocol described by (Wu and Letchworth, 2004) with the following modifications: 2 μg of linearized vector DNA were used per transformation. After electroporation by pulse at 1.5 kV in a 2 mm electroporation cuvette, cells were allowed to recover in YPDS medium for 1 hour at 30° C. The selection was done directly for resistance to 1 mg/ml of G418 on YPDS plates for 60-80 hours.

[0377] Resulting clones were screened for expression of a desired protein by inoculating a single colony into BMMY medium in a well of a deep-well plate and incubating at 28° C. shaking at 500 rpm for 48 hours and analysing the resulting culture medium by SDS-PAGE. The best clones were used for medium scale expression by growing a culture of the desired strain in BMGY at 28° C. shaking at 120 rpm to mid-log phase, harvesting the cells and resuspending them in BMMY to induce expression under control of AOX1 promoter and incubating a culture at 28° C. shaking at 120 rpm. Additional methanol was supplied 24 hours post-induction to replenish consumed/evaporated methanol. After 48 hours post-induction, cells were removed from the culture by centrifugation at 3000 g for 10 minutes, then at 10000 g for 10 minutes. The final supernatant was filtered through a 0.2 μm filter before proceeding to VHH purification.

[0378] Example 8—Highly Potent Neutralizers of the South African B.1.351 SARS-CoV-2 Strain (Beta Variant)

[0379] In a further set of experiments, the inventors assessed the neutralization potency of their mutation-tolerant VHH antibodies, using the South African B.1.351 variant as a representative of the recently emerged mutant strains. As their microscopic assay relied on VHH antibody-based detection of newly made viral proteins, they had to ensure that the mutant spike also yields an unambiguous signal. This was achieved with a mix of VHH-antibodies against epitopes outside the RBD (anti-S1ΔRBD), against the non-mutated epitope 2 (Re7E02 or Re9C07), and by the mutation-tolerant main epitope-binder Re9H03.

[0380] By contrast, Re6D06, which fails in mutant RBD binding, also failed to stain mutant spikes of infected cells suggesting that combinations of mutation-sensitive and—tolerant VHH-antibodies can diagnose virus variants by simple staining procedures.

[0381] The inventors then compared two strategies for the actual neutralization of mutant B.1.351 (FIG. 9). They first tested the most promising VHH monomers and observed potent neutralization by Re5F10 (at 1.7 nM), Re6H06 (at 170 pM), Re9B09 (at 1.7 nM), and by the mutant-preferring Re9B09 class member Re9H03 (at 50-170 pM; FIG. 9A, Table 5). Even more potent B.1.351 neutralization was found for the heterodimers Re9F06-R28 (50 pM), Re9F06-Re9B09 (50 pM), and Re9F06-Re6H06 (17 pM; FIG. 9B), as well as for the analogous Re21D01-heterodimers with R28, Re9B09 and Re6H06, whereby VHH-antibody-connecting linkers of 14, 15, or 29 amino acids gave rather similar results (for additional data, see Table 5 for VHH monomers and Table 6 for VHH heterodimers).

TABLE-US-00007 TABLE 5 Lowest concentration VHH monomer SEQ ID neutralizing B.1.351 (nM) Re5F10 29 1.7-5 Re21D01 (Re5F10 class) 260 5 Re26E09 (Re5F10 class) 284 1.7-5 Re26E11 (Re5F10 class) 288 1.7-5 Re21H01 5 Re6H06 81 0.170 Re26D07 (Re6H06 class) 280 0.500 Re9B09 129 1.7 Re9H03 (Re9B09 class) 252   0.050-0.170 Neutralization of the South African B.1.351 SARS-CoV-2 variant by mutation-tolerant VHH antibodies

TABLE-US-00008 TABLE 6 Lowest neutralizing concentration (pM) Alpha Beta Wild UK South African VHH1 Spacer VHH2 Expression type B.1.1.7 B.1.351 Re9F06 A Re9B09 E.coli 50-170 17-170 Re9F06 A Re5D06R28D E.coli  5-50  5-170 Re22D04 A Re5D06R28D E.coli 170 Re25H10 A Re9B09 E.coli 500 50-170 170 Re25H10 A Re5D06R28D E.coli 170 170 170 Re9F06 B Re5D06R28D Pichia  50 Re9F06 C Re6H06 Pichia 500  50 170 Re9F06 C Re9B09 Pichia 17-170 170 50-170 Re9F06 B Re6H06 Pichia 17-170  50 Re9F06 B Re9B09 Pichia 50-170 17-170 Re9F06 B Re5D06R15_3QE Pichia 5-50 50-170 Re9F06 B Re5D06R28_3QE Pichia 50-170 Re21D01 B Re5D06R15_3QE Pichia 50-170 Re21D01 B Re5D06R28_3QE Pichia 17-50 50-170 Re21D01 C Re5D06R15_3QE Pichia  50 170 Re21D01 C Re5D06R28_3QE Pichia  50  50 Re21D01 B Re6H06 Pichia  50 50-500 Re21D01 B Re9B09 Pichia  50 Re21D01 C Re6H06 Pichia 170 50-500 50-170 Re21D01 C Re9B09 Pichia 50-170 170 Neutralization of the South African B.1.351 SARS-COV-2 variant by VHH-spacer-VHH tandem fusions. Spacer A: TSGEGEGGEGGEGS (SEQ. ID NO: 312); Spacer B: TSGEGEGGGEGGEGS (SEQ. ID NO: 313); Spacer C: TSGEGEGGGEGGSGEGGSEGGEGGSGEGS (SEQ. ID NO: 314).

Example 9: Binding of Mutation-Tolerant Monomeric VHHs and Heterodimeric VHHs to the RBD of the SARS-CoV-2Delta Variant

[0382] In further experiments, the inventors used Biolayer interferometry (BLI) to assess the binding strength of selected VHHs to the receptor-binding domain of the Delta variant of SARS-CoV-2, which carries the L452R and T478K mutations. For this, VHH constructs were labelled with Biotin-PEG.sub.11-Maleimide (#PEG1595; Iris Biotech) through N- and C-terminally introduced cysteines.

[0383] BLI experiments were performed using High Precision Streptavidin biosensors and an Octet RED96e instrument (ForteBio/Sartorius) at 25° C. with Phosphate-Buffered Saline (PBS) pH 7.4, 0.02% (w/v) Tween 20 and 0.1% (w/v) BSA as assay buffer.

[0384] The indicated VHHs were immobilized on sensors until a binding signal of 0.4 nm (for monomeric VHHs) or 0.75 nm (for heterodimeric VHHs) was reached. Subsequently, the biosensors were dipped into wells containing 3-fold dilutions (20, 6.66, and 2.22 nM) of the wild type RBD (Z03479, GenScript) or the B.1.617.2/Delta RBD (40592-V08H90, Sino Biologicals) for 450 s. RBD dissociation in assay buffer was followed for 900 s. Data were reference-subtracted and curves were fitted using a mass transport model (Octet Data Analysis HT 12.0 software). The dissociation constants (K.sub.Ds) are summarized in the Table shown below. They revealed very tight binding of the analyzed VHHs and heterodimeric VHHs.

TABLE-US-00009 TABLE 7 K.sub.D RBD K.sub.D RBD VHH monomer or VHH heterodimer SEQ ID Wild type Delta variant Re5F10 29    40 pM 40 pM Re6H06 81 ≤10 pM ≤10 pM Re9B09 129 ≤10 pM 100 pM Re5D06R15 224 ≤10 pM 30 pM Re5D06R28 232 ≤10 pM 80 pM Re9F06-spacerA-Re9B09 292 ≤10 pM ≤10 pM Re9F06-spacerA-Re5D06R28D 293 ≤10 pM ≤10 pM

Example 10: Neutralization of the SARS-CoV-2 Delta Variant by Mutation-Tolerant VHHs and VHH Tandems

[0385] The indicated VHH constructs were used in neutralization experiments of the Delta SARS-CoV-2 variant. Experiments were performed analogously to those described in FIGS. 5 and 9. Table 8 lists the lowest neutralizing concentrations as geometric means of three independent experiments.

TABLE-US-00010 TABLE 8 Lowest neutralizing SEQ concentration VHH monomer or VHH heterodimer ID Delta variant (nM) Re6H06 81 0.5 Re9B09 129 1.7 Re9F06-SpacerB-Re5D06R28D 297 0.05 Re9F06-SpacerB-Re5D06R15_3QE 302 0.09 Re9F06-SpacerB-Re5D06R28_3QE 303 0.09 Re21D01-SpacerC-Re5D06R28_3QE 307 0.29 Re21D01-SpacerB-Re5D06R28_3QE 305 0.29 Re9F06-SpacerC-Re9B09 299 0.29 Re9F06-SpacerB-Re6H06 300 0.50 Re9F06-SpacerC-Re6H06 298 0.50 Re21D01-SpacerC-Re6H06 310 0.50

Example 11: The Trimerization Module of Collagen XVIII Allows for More Potent SARS-CoV-2 Neutralizing VHH Fusions than the Homologous NC1 Domain of Collagen XV

[0386] The indicated trimeric VHH fusions were expressed in E. coli, purified and tested in a SARS-CoV-2 neutralization assay as described in FIGS. 3 and 5.

[0387] The lowest neutralizing concentrations (referring to VHH moieties) are listed in Table 9. The collagen XVIII NC1 domain was used as a C-terminal fusion partner. The collagen XV NC1 domain was used an N-terminal fusion partner in Re9B09, Re9H01 and Re7H02 (Re6B06 class) fusions, and as a C-terminal fusion partner in the KG4B11 fusion.

[0388] The data shows that the C-terminal collagen XVIII fusion allows for greater potency than an collagen XV NC1 on either the N-terminus or the C-terminus.

TABLE-US-00011 TABLE 9 Collagen XV Collagen XVIII NC1 fusion NC1 fusion Lowest Lowest neutralizing neutralizing SEQ concentration SEQ concentration VHH trimer ID (pM) ID (pM) Re9B09 315 90 316 16 Re9H01 317 50 318 4 Re7H02 (Re6B06 class) 319 160 212 4 KG4B11 (Re6B06 class) 320 170 213 4

[0389] Further examples are detailed in the figures and figure legends.

REFERENCES

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